Sugar, which is one of biomolecule that is the most widely and abundantly present in nature, is the most common molecule involved in recognition and signaling in cell. Most sugar constituent units may be added to the aglycon, when being activated by nucleic acids. Nucleotide-sugar has an activated form of monosaccharide, and serves as a donor in a transglycosylation reaction by glycosyltransferase. However, the transglycosylation still has problems in that reactivity of the glycosyltransferase is weak, utilization of activated glycan constituent units is limited, and the like.
Recently, as research into a structure and function of sugar chain has been rapidly conducted, usage development of the sugar chain as drug or functional material including oligosaccharide, sugar lipids, glycoproteins, and the like, having physiological activities has been actively conducted. Among them, a sialic acid-containing sugar chain containing N-acetylneuraminic acid (NeuAc) at the end is a sugar chain having important functions as cell adhesion or a role as an acceptor in virus infection, and the like.
The sialic acid-containing sugar chain is generally synthesized by catalysis of sialyltransferase. The sialyltransferase is an enzyme transferring sialic acid to acceptors such as a sugar chain, and the like, using CMP-N-acetylneuraminic acid as a sugar supplier. However, practically, CMP-N-acetylneuraminic acid used as the sugar supplier is significantly expensive and only a small amount corresponding to a reagent level is supplied.
As a method for preparing CMP-N-acetylneuraminic acid, a synthesis method by CMP-N-acetylneuraminic acid synthetase enzyme, using cytidine 5′-triphosphate (CTP) and N-acetylneuraminic acid (NeuAc) as substrates, has been known. However, since CTP and NeuAc are expensive raw materials, CMP-N-acetylneuraminic acid to be synthesized by directly using the raw materials is also expensive.
As a method for preparing CMP-N-acetylneuraminic acid (CMP-NeuAc), the following methods have been reported: (1) a method for preparing CMP-NeuAc from N-acetyl-D-mannosamine (ManNAc) using N-acetylneuraminic acid lyase or N-acetylneuraminic acid synthetase (J. Am. Chem. Soc., 110:6481, 1988; J. Am. Chem. Soc., 110:7159, 1988; Japanese Patent Laid-Open Publication No. Hei 10-4961), (2) a method for preparing N-acetylneuraminic acid (NeuAc) by converting N-acetyl-D-glucosamine (GlcNAc) into N-acetyl-D-mannosamine (ManNAc) under alkaline conditions and adding N-acetylneuraminic acid lyase or N-acetylneuraminic acid synthetase thereto (Japanese Patent Laid-Open Publication No. Hei 5-211884; Biotechnol. Bioeng., 66:2, 1999; Enzyme Microb. Technol., 20, 1997), (3) a method for preparing NeuAc from N-acetyl-D-glucosamine (GlcNAc) using N-acetylglucosamine (GlcNAc) 2-epimerase catalyzing conversion from GlcNAc into ManNAc, N-acetylneuraminic acid lyase or N-acetylneuraminic acid synthetase (WO 95/26399; Japanese Patent Laid-Open Publication No. Hei 3-180190; Japanese Patent Laid-Open Publication No. 2001-136982), (4) a method for synthesizing CMP-N-acetylneuraminic acid using E. coli and yeast cells, and the like.
However, the method (1) has a problem in that N-acetyl-D-mannosamine (ManNAc) is expensive raw material, and the method (2) has a problem in that a process for purifying ManNAc from a mixture of GlcNAc and N-acetyl-D-mannosamine (ManNAc) is extremely complicated even though N-acetyl-D-glucosamine (GlcNAc) is a cheap raw material. In addition, since GlcNAc2-epimerase used in the method (3) requires ATP (adenosine triphosphate), the method (3) has problems in that it is required to add expensive ATP or produce ATP from ATP precursor, adenine, using microorganism. The method (4) has a problem in that the use of Escherichia coli (E. coli) and yeast cells is complicated in view of process.
Korean Patent Laid-Open Publication No. 10-2006-0010706 discloses a method for preparing CMP-N-acetylneuraminic acid by adding cytidine 5′-monophosphate (CMP), N-acetyl-D-glucosamine, pyruvate (sodium pyruvate) and a yeast to a transformant into which a co-expression vector including a gene encoding N-acetylglucosamine-2-epimerase and a gene encoding N-acetylneuraminic acid aldolase is introduced to thereby synthesize neuraminic acid, and further adding a CMP-N-acetylneuraminic acid synthetase, or adding cytidine 5′-monophosphate (CMP), N-acetyl-D-glucosamine, pyruvate and a yeast to a transformant into which a co-expression vector including a gene encoding N-acetylneuraminic acid aldolase and a gene encoding CMP-N-acetylneuraminic acid synthetase is introduced. However, the method has problems in that various steps need to be performed in preparing CMP-N-acetylneuraminic acid, and a conversion yield from cytidine 5′-monophosphate (CMP) used as a substrate into cytidine 5′-triphosphate (CTP) is low.
Glycans including sialyl oligosaccharides and fucose in glycoproteins and glycolipids play a significantly important role in biological processes in various ways.
However, the conventional known reactions for combining sialic acid with derivatives of biologically active materials have disadvantages in that the sialyl acid derivatives are prepared by sialyltransferase, using expensive CMP-N-acetylneuraminic acid as a starting material, and the preparation efficiency is also low. Further, technology for preparing sialic acid derivative using N-acetyl-D-glucosamine as a starting material has disadvantages in that preparation efficiency of the sialic acid derivative is low since activity range and activity of the sialyltransferase are reduced (Kim, Dae-Hee, Sun Moon graduate School of Science doctoral dissertation, 2011).
Accordingly, the present inventors made an effort to develop a method for preparing a sialic acid derivative of a biologically active material at a high efficiency and a low cost, found that when a step of preparing CMP-N-acetylneuraminic acid using N-acetyl-D-glucosamine and cytidine 5′-monophosphate (CMP) as starting materials and a step of preparing a biologically active material derivative combined with sialic acid from the prepared CMP-N-acetylneuraminic acid are performed in a single reactor, using a sialyltransferase mutant, cytidine 5′-monophosphate (CMP) which is expensive raw material, is capable of being recycled, and a sialic acid derivative of the biologically active material is capable of being prepared at a high yield, and completed the present invention.